Legionella longbeachae effector protein RavZ inhibits autophagy and regulates phagosome ubiquitination during infection

Legionella organisms are ubiquitous environmental bacteria that are responsible for human Legionnaires’ disease, a fatal form of severe pneumonia. These bacteria replicate intracellularly in a wide spectrum of host cells within a distinct compartment termed the Legionella-containing vacuole (LCV). Effector proteins translocated by the Dot/Icm apparatus extensively modulate host cellular functions to aid in the biogenesis of the LCV and intracellular proliferation. RavZ is an L. pneumophila effector that functions as a cysteine protease to hydrolyze lipidated LC3, thereby compromising the host autophagic response to bacterial infection. In this study, we characterized the RavZ (RavZLP) ortholog in L. longbeachae (RavZLLO), the second leading cause of Legionella infections in the world. RavZLLO and RavZLP share approximately 60% sequence identity and a conserved His-Asp-Cys catalytic triad. RavZLLO is recognized by the Dot/Icm systems of both L. pneumophila and L. longbeachae. Upon translocation into the host, it suppresses autophagy signaling in cells challenged with both species, indicating the functional redundancy of RavZLLO and RavZLP. Additionally, ectopic expression of RavZLLO but not RavZLP in mammalian cells reduces the levels of cellular polyubiquitinated and polyneddylated proteins. Consistent with this process, RavZLLO regulates the accumulation of polyubiquitinated species on the LCV during L. longbeachae infection.


Introduction
Legionella are gram-negative bacteria ubiquitously found in natural environments [1]. When contaminated aerosols are inhaled by humans, Legionella can cause a severe form of pneumonia termed Legionnaires' disease, which can be fatal if not promptly and appropriately treated [1]. To date, more than 60 Legionella species have been recognized, and almost half of them have been reported to cause human disease [2]. Legionella pneumophila and Legionella longbeachae are the most frequently isolated species in cases of Legionnaires' disease [2]. L. in L. longbeachae biology and infection processes. The only example is the effector protein SidC, which functions similarly to its homolog in L. pneumophila to promote the interaction of the LCV with the ER [26]. Despite the translocation of a distinct set of effectors, a recent study illustrated that both L. pneumophila and L. longbeachae develop similar replicative vacuoles in infected cells, albeit through different mechanisms [27]. Autophagy is a cellular process that occurs in eukaryotic cells and targets cytosolic proteins, lipids and organelles to lysosomes for degradation. During the process, sequestered cargoes are enclosed by a membrane-bound compartment termed the autophagosome [28]. Following fusion with a lysosome, the cargoes are consumed in the autolysosome [28]. Autophagy can be a nonselective process that degrades cytoplasmic components to offset the effects of starvation [29]. In contrast, selective autophagy targets specific substrates for degradation and is divided into different types according to the sequestered cargo (e.g. mitophagy, pexophagy, reticulophagy and xenophagy) [29]. Xenophagy is a host autophagic response for the degradation of invading microbes and is recognized as a conserved host immune response against intracellular pathogens [30]. Moreover, many successful intracellular bacteria have evolved distinct mechanisms to avoid clearance by xenophagy [31]. For example, L. pneumophila utilizes the effector protein RavZ, a cysteine protease, to inhibit autophagy through the irreversible deconjugation of LC3 from phosphatidylethanolamine (PE) [32]. To date, the host autophagic response to L. longbeachae infection has not been reported. In this study, we found that the RavZ ortholog in L. longbeachae RavZ LLO plays important roles in the inhibition of the host autophagy pathway. In addition, RavZ LLO decreases cellular polyubiquitin levels when ectopically expressed in mammalian cells and regulates the association of polyubiquitinated species with the LCV in L. longbeachae-infected cells.

Bacterial strains, plasmids, and growth conditions
The strains, plasmids, and primers used in this study are provided in S1-S3 Tables, respectively. Escherichia coli strains were grown in Luria-Bertani (LB) medium or LB agar plates. When needed, ampicillin (100 μg/mL), kanamycin (30 μg/mL), or chloramphenicol (30 μg/ mL) was added to the E. coli cultures. L. pneumophila (Lp02), L. longbeachaea ATCC33462, and their derivatives were cultured on charcoal yeast extract (CYE) agar plates or in N-(2-acetamido)-2-aminoethanesulfonic acid-buffered yeast extract broth (AYE) at 37˚C. If needed, antibiotics were supplemented to the Legionella cultures at the following concentrations: ampicillin (100 μg/mL), kanamycin (20 μg/mL), streptomycin (50 μg/mL), and chloramphenicol (5 μg/mL). When necessary, thymidine was added at 200 μg/mL. The in-frame deletion mutants L. pneumophila ΔravZ, L. longbeachae ΔravZ LLO , and L. longbeachae ΔdotB were generated by allelic exchange as described previously [33,34]. To determine the translocation of RavZ LLO , fragments amplified from L. longbeachae genomic DNA were inserted into pXDC61m [35], and the resulting plasmid was introduced into relevant Legionella strains by electroporation (2.5 kV; 200 O; 0.25 μF). pXDC61JQ was modified based on the backbone of pDXC61m and used for IPTG-inducible expression of Flag-tagged proteins in L. longbeachae. To achieve this goal, we inserted a Nde-Flag-BamH-Bgl-Sac-Xho-Sal-Hind polylinker into Nde/HindIII-digested pDXC61m to remove the TEM gene. The ravZ LLO gene was then cloned into pXDC61JQ and electroporated into L. longbeachae strains as described above. For complementation of L. pneumophila strains, ravZ LP and ravZ LLO were inserted into pZL507 [36] and similarly transformed into L. pneumophila. For the expression of proteins in mammalian cells, amplified DNA products were cloned into pCMV-4×Flag [37] or peGFPC1 by standard cloning methods. For protein expression in E. coli, genes were inserted into pET28a.
Site-directed mutagenesis of ravZ LLO was performed by the Quikchange kit (Agilent) with primers designed by the QuikChange Primer Design program (https://www.agilent.com.cn/ store/primerDesignProgram.jsp). The integrity of all plasmids was confirmed by DNA sequencing.

Recombinant protein purification and in vitro deubiquitination assay
E. coli BL21 (DE3) harboring pET28-RavZ was cultured to an OD 600 of 0.6-0.8 at 37˚C in LB broth. After isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, the culture was further incubated in a shaker (220 rpm/min) at 16˚C for 16-18 h. The bacterial cells were collected by centrifugation and resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0). Cells were lysed twice using a JN-Mini Low Temperature Ultrahigh Pressure Continuous Flow Cell Cracker (JNBIO, Guangzhou, China). Cell lysates were cleared by centrifugation at 12000×g for 20 min. The supernatant containing the protein of interest was mixed with Ni 2+ -NTA beads and incubated at 4˚C for 1 h with end-to-end rotation. The beads were washed with 10 bead volumes of washing buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0), and 6×His-tagged proteins were eluted with elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0). Eluted proteins were dialyzed twice to remove imidazole in a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl and 10% (v/v) glycerol. Protein concentration was measured by the Bradford assay using BSA levels for normalization.
During the diubiquitin cleavage assay, 1 μM purified His 6 -RavZ LLO was incubated with each diubiquitin (1 μM, Boston Biochem) in 20 μL DUB buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT). The reaction was allowed to proceed for 2 h at 37˚C and was stopped by the addition of 5×SDS loading buffer. The cleavage of diubiquitin was analyzed by Coomassie brilliant blue staining.
To determine the deubiquitination or deneddylation activity of RavZ in cells, HA-Ub or Flag-Nedd8 was cotransfected with GFP-RavZ LP , GFP-RavZ LLO or GFP-RavZ LLOC251A into HEK293T cells. GFP-SdeA Dub and GFP-SENP8 were used as positive controls for deubiquitination and deneddylation, respectively. Twenty-four hours after transfection, transfected cells were harvested and lysed in NP40 lysis buffer for 10 min on ice. Then, the lysates were centrifuged at 12,000×g at 4˚C for 10 min to remove insoluble fractions. Polyubiquitinated and polyneddylated species were enriched by anti-HA and anti-Flag agarose beads at 4˚C for 8 h on a rotatory shaker, respectively. After washing 3 times with lysis buffer, the beads were resuspended in 50 μL of 1×SDS loading buffer and boiled for 5 min at 100˚C.
To detect the cellular localization of RavZ LLO , HeLa cells were transfected with GFP-RavZ LLO for 24 h. Samples fixed with 4% paraformaldehyde were immunostained with organelle-specific primary antibodies. To investigate the influence of RavZ on LC3 puncta formation under transfection conditions, HEK293 cells were cotransfected with GFP-LC3 and 4×Flag-tagged RavZ LP , RavZ LLO or RavZ LLOC/A for 24 h. After the cells were fixed with 4% paraformaldehyde, the percentage of LC3 puncta was measured under a fluorescence microscope.

Bacterial infections
During infection experiments, overnight liquid cultures of stationary-phase Legionella bacteria (OD 600 = 3.3-3.8) were used to infect cells at the indicated MOIs. To evaluate the translocation of RavZ LLO , RAW264.7 cells seeded in 96-well plates at a density of 1×10 6  To assess the intracellular replication of L. longbeachae, U937-derived macrophage cells seeded in 24-well plates were challenged with WT L. longbeachae, ΔdotB, and ΔravZ LLO at an MOI of 10 in triplicate. At 2 h post-incubation, extracellular bacteria were removed by washing the samples with warm PBS three times. After fresh culture medium was added, the infections were allowed to proceed for 24, 48, and 72 h. Cells were collected at each assay time point and lysed with 0.2% saponin. Serial dilutions of the lysates were plated on CYE plates and grown for 4 days at 37˚C before the CFUs were enumerated.
To inspect LCV-associated RavZ LLO or polyubiquitinated species, 4×10 5 PMA-differentiated U937 cells seeded on glass coverslips in 24-well plates were infected with relevant Legionella strains at an MOI of 10 for 2 h. After they were extensive washed with PBS, samples fixed with 4% paraformaldehyde were immunostained using the indicated antibodies specific for L. longbeachae, Flag, or polyubiquitin.
To assess the impact of RavZ on LC3-II levels in infected cells, HEK293 cells transfected to express the FcγII receptor were infected with the indicated opsonized Legionella strains at an MOI of 50 for 2 h in the presence of 160 nM bafilomycin A1 (Sigma) as described earlier.
Lysates were prepared by lysing the cells with NP40 lysis buffer, and the level of LC3-II was measured by immunoblotting analysis. To assess the formation of LC3 puncta in infected cells, A549 cells were transfected with mCherry-LC3 and FcγII receptor. At 24 h post-transfection, anti-Legionella antibody opsonized bacteria were added to the cells supplemented with 160 nM bafilomycin A1. After 2 h of infection, samples fixed with 4% paraformaldehyde were immunostained with the appropriate antibodies.
During the immunostaining experiments, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature (RT). After permeabilization with 0.2% Triton X-100 for 5 min, the samples were stained with the appropriate antibodies for 1 h at RT. Rat and rabbit anti-L. longbeachae antibodies were commercially generated by AbMax Biotechnology Co. (Beijing, China) and diluted at 1:2,000 for immunostaining. Other primary antibodies used in this study were as follows: anti-L. pneumophila (1:10,000), anti-Flag (Sigma, cat# F1804, 1:200), anti-FK1 (Enzo, cat# BML-PW8805, 1:1,000), and anti-calnexin (Abcam, ab22595, 1:100). After the samples were washed with PBS 3 times, they were incubated with appropriate secondary antibodies conjugated to specific fluorescent dyes for 1 h at RT. Fluorescent images were acquired by an Olympus IX-83 fluorescence microscope.

Data analysis
ImageJ was used to quantify protein abundance. Prism 8.0 (GraphPad, USA) with two-tailed Student's t test was used to analyze the data. Results with P values less than 0.05 were considered significant.

RavZ LLO is translocated into host cells by the Dot/Icm secretion system
L. pneumophila effector protein RavZ is a cysteine protease that functions to inhibit the host autophagy response by the deconjugation of lipidated LC3 [32]. Genes coding for RavZ are present in 4 out of the 41 sequenced genomes covering 38 Legionella species [23]. Although more than 66% of L. pneumophila effectors are missing in L. longbeachae [4], the RavZ ortholog exists in L. longbeachae and is encoded by llo_2508 (ravZ LLO ). RavZ LLO shares 59.4% overall sequence identity with RavZ from L. pneumophila, and the catalytic cysteine residue for the enzymatic activity is conserved between the two proteins (S1 Fig). To determine whether RavZ LLO is a bona fide Dot/Icm substrate of L. longbeachae, we employed the β-lactamase reporter assay that has been successfully applied to identify and validate effector proteins of Legionella spp. [27,35,39]. Infection of RAW264.7 cells with L. longbeachae producing TEM--RavZ LLO led to obvious translocation of the fusion protein, which was evidenced by 27% of the cells showing blue fluorescence signals emitted by the β-lactamase substrate CCF4-AM ( Fig  1A). In addition, samples infected by the wild-type L. pneumophila strain harboring TEM--RavZ LLO displayed blue cells at a proportion of 35% (Fig 1A). In contrast, despite producing considerable levels of TEM-RavZ LLO , translocation of the fusion protein did not occur when cells were challenged with the dot/icm-deficient strains (Fig 1A and 1B). Taken together, our data demonstrated that RavZ LLO is an effector protein recognized by the Dot/Icm machinery of both L. longbeachae and L. pneumophila.

RavZ LLO is associated with the LCV and is dispensable for the intracellular growth of L. longbeachae
To test whether RavZ LLO is targeted to any specific organelles in human cells, we generated green fluorescence protein (GFP)-tagged RavZ LLO and ectopically expressed the construct in HeLa cells by transfection. The subcellular localization of RavZ LLO was analyzed under fluorescence microscopy after cells were immunostained with antibodies specific for different organelle markers. GFP-RavZ LLO expression showed extensive overlap with the ER-resident protein calnexin, suggesting that RavZ LLO is targeted to the ER (Fig 2A).
Next, we investigated the cellular distribution of RavZ LLO during infection. We generated a plasmid bearing Flag-tagged RavZ LLO and transformed it into both WT and dotBmutant L. longbeachae strains. U937 cells were then infected with these strains and immunostained with Flag antibodies. After 2 h of infection, Flag-RavZ LLO staining signals were detected in 84% of the LCVs harboring WT L. longbeachae (Fig 2B-2D). In contrast, despite similar Flag-RavZ LLO expression levels, few LCVs were positively stained with Flag antibody when the cells were infected with the L. longbeachae strain lacking a functional Dot/Icm system (Fig 2B-2D). These data indicate that RavZ LLO is associated with the LCV after being translocated into infected cells by L. longbeachae.
To characterize the role of RavZ LLO in bacterial infection, we first made a ravZ LLO deletion mutant of the L. longbeachae strain by allelic exchange. Then, the intracellular replication of the mutant strain was monitored in U937 cells. After 3 days of infection, approximately 3 orders of magnitude more WT L. longbeachae were recovered, whereas strains with the absence of dotB failed to replicate in the host cells ( S2 Fig). Notably, the deletion of ravZ LLO in L. longbeachae did not cause a significant growth defect at any assayed time point (S2 Fig). These results suggest that ravZ LLO is dispensable for the intracellular proliferation of L. longbeachae.

Ectopic expression of RavZ LLO in mammalian cells inhibits host autophagy
Considering that RavZ inhibits the host autophagy response and the high sequence similarity between RavZ LP and RavZ LLO , we reasoned that RavZ LLO and RavZ LP may exhibit identical biological functions. To this end, we cotransfected HeLa cells with GFP-LC3 and Flag-tagged RavZ, and the formation of LC3 puncta was measured under a fluorescence microscope. In the absence of RavZ, 75% of the cells showed punctate LC3-positive autophagosomes (APs), which was a result of basal levels of autophagy (Fig 3A-3C). In contrast, the percentage of cells with LC3 puncta was reduced to 9% when RavZ LP was coexpressed (Fig 3A-3C). Similarly, the number of LC3 puncta-containing cells was significantly decreased in the presence of RavZ LLO (Fig 3A-3C). Additionally, mutation of Cys251 to Ala in RavZ LLO remarkably alleviated its ability to inhibit host autophagy (Fig 3A-3C).
RavZ behaves as a cysteine protease that irreversibly uncouples PE-conjugated LC3 on AP membranes. Therefore, we further evaluated RavZ LLO -mediated autophagy evasion by measuring the levels of the lipidated form of LC3 (LC3-II) by Western blot analysis. Notably, although less prominent than RavZ LP expression, the expression of GFP-RavZ LLO indeed significantly reduced endogenous LC3-II levels in cells treated with bafilomycin A1 (Fig 3D  and 3E). The cysteine protease activity of RavZ LLO was responsible this decrease since the expression of the catalytically inactive mutant resulted in a considerable level of LC3-II as the GFP control (Fig 3D and 3E).

RavZ LLO inhibits autophagy in Legionella infection
Having proven the functional role of RavZ LLO in autophagy inhibition in transfected cell lines, we further investigated its biological relevance in L. longbeachae infection. To this end, we infected HEK293 cells with L. longbeachae strains and monitored the LC3-II levels. Similar to L. pneumophila, samples infected with the WT L. longbeachae strain showed a significant blockage of LC3-II generation (Fig 4A and 4B). However, the LC3-II level was not affected when the cells were challenged with the dotB deletion mutant, suggesting Dot/Icm-dependent inhibition of the host autophagy system by L. longbeachae (Fig 4A and 4B). Consistent with this observation, infection of A549 cells stably expressing mCherry-LC3 with virulent L. longbeachae led to a considerably lower percentage of cells containing punctate LC3-positive APs than infection with the Dot/Icm-deficient mutant (Fig 4C and 4D). The autophagy inhibition that is mediated by L. longbeachae could be attributed to the activity of RavZ LLO because a similar level of LC3-II and percentage of punctate LC3-positive APs in infected cells were detected in samples receiving L. longbeachae ΔdotB and ΔravZ LLO (Fig 4A-4E). Autophagy inhibition was completely restored by the introduction of a plasmid encoding ravZ LLO into the ΔravZ LLO mutant (Fig 4A-4E). However, complement of the mutant strain with catalytically inactive RavZ LLO failed to suppress the autophagic response in either assay (Fig 4A-4E). Hence, RavZ LLO is necessary to block autophagy during L. longbeachae infection.
Since RavZ LLO was also recognized by the L. pneumophila Dot/Icm system, we analyzed whether the defect in autophagy inhibition displayed by L. pneumophila ΔravZ could be complemented by RavZ LLO . Consistent with previous observations [32], the ΔravZ mutant and ΔdotA mutant of L. pneumophila were equally defective in autophagy inhibition, which was determined by the levels of lipidated LC3 as well as the proportion of LC3 puncta-positive cells (Fig 5A-5E). Interestingly, both phenotypes were fully restored by the production of not only RavZ LP but also RavZ LLO from plasmid-borne genes (Fig 5A-5E). Taken together, our data

Expression of RavZ LLO in mammalian cells decreases cellular polyubiquitination and polyneddylation
Previous structural studies showed that the N-terminal domain of RavZ harbors a fold similar to that of the Ubl-specific protease (Ulp) family deubiquitinase (DUB) [40]. In addition, bioinformatics analysis of both RavZ LP and RavZ LLO using HHpred revealed distant homology of these proteins with known bacterial DUBs, including SseL from Salmonella enterica serovar Typhimurium (S4 Table). The His-Asp-Cys catalytic triad critical for SseL function is conserved in RavZ LLO (S3 Fig). These observations together illustrate the potential role of RavZ LLO in hydrolyzing polyubiquitin chains. To test this hypothesis, we cotransfected HEK293T cells with GFP-RavZ and human influenza hemagglutinin (HA)-tagged ubiquitin and assayed the polyubiquitinated protein levels by immunoblotting. In vivo production of RavZ LLO but not RavZ LP significantly reduced the levels of cellular polyubiquitinated species, which was shown by the decreased expression of the high-molecular-weight ubiquitin ladder detected by the anti-HA antibody (Fig 6A). Surprisingly, the substitution of Cys251 to Ala led to a similar decrease in polyubiquitin signals as that observed in the WT RavZ LLO (Fig 6A). Similarly, ectopic expression of RavZ LLO but not RavZ LP decreased cellular polyneddylation, a ubiquitin- like modification, in a Cys251-independent manner (S4 Fig). These data indicate that RavZ LLO can cleave polyubiquitin and polynedd8 chains, and the catalytic cysteine responsible for deconjugating LC3-PE is not important for such activity.
Next, we aimed to characterize the DUB activity of RavZ LLO in in vitro biochemical reactions using a panel of diubiquitins linked by different lysine residues. However, recombinant RavZ LLO obtained from E. coli failed to hydrolyze any of the tested diubiquitins ( Fig 6B). Therefore, it is possible that RavZ LLO requires a cofactor to activate its DUB activity, as it is ectopically expressed in mammalian cells. Alternatively, RavZ LLO might activate the DUB activity of host proteins to decrease cellular polyubiquitination and polyneddylation.

RavZ LLO reduces the association of polyubiquitinated species with the LCV
During L. pneumophila infection, the LCVs are known to be decorated with polyubiquitinated protein by a process depending on the Dot/Icm system [41]. Despite the lack of established DUB activity, RavZ could interfere with ubiquitin accumulated on Salmonella-containing vacuoles in a Legionella and Salmonella coinfection system [42]. However, RavZ did not affect ubiquitin recruitment to LCVs, which may be attributed to an unknown antagonistic mechanism mediated by Legionella [42]. Considering the polyubiquitin deconjugating activity of RavZ LLO in mammalian cells and its localization on bacterial phagosomes during L. longbeachae infection, we speculated that this protein might play a role in the regulation of ubiquitin association on the LCV. To test this hypothesis, we infected U937 cells with relevant L. longbeachae strains followed by immunostaining with an FK1 antibody that specifically recognizes ubiquitinated proteins. Similar to L. pneumophila infection, vacuoles harboring virulent L. longbeachae were also enriched with polyubiquitinated species, and 66% of the LCVs were positively stained with FK1 antibodies at 2 h post-infection (Fig 7A and 7B). Importantly, the absence of ravZ LLO resulted in a significant increase in the proportion of ubiquitin-positive vacuoles (Fig 7A and 7B). Moreover, expression of RavZ LLO from a plasmid in the L. longbeachae ΔravZ LLO mutant restored ubiquitin association with the LCV (Fig 7A and 7B). Interestingly, although challenging U937 cells with the L. pneumophila strain lacking the ravZ gene was able to promote the recruitment of ubiquitinated proteins to the bacterial phagosomes as efficiently as challenge with the WT L. pneumophila, complement of the L. pneumophila ΔravZ mutant with a plasmid-borne ravZ LLO appeared to considerably decrease the percentage of ubiquitin-positive LCVs (Fig 8A and 8B). These results suggest that RavZ LLO contributes to the removal of ubiquitin from bacterial phagosomes.

Discussion
The host autophagy pathway not only plays vital roles in maintaining intracellular homeostasis but can also serve as an important cell autonomous immune response that eliminates intracellular bacteria to counteract infection [43]. As a successful intracellular pathogen, L. pneumophila has developed various effector-driven mechanisms that may interplay to promote the evasion of bacterial clearance by the host xenophagic response. These strategies include the prevention of autophagosome formation via cleavage of LC3-PE by RavZ [32], disturbance of sphingolipid biosynthesis through the Legionella effector sphingosine-1-phosphate lyase (LpSpl) [44], and degradation of syntaxin 17 via the serine protease activity of effector Lpg1137 [45]. Moreover, a panel of effector proteins could indirectly impact host autophagy through interference with mTORC1 signaling. The SidE effector family and SetA are inhibitors of mTORC1 that can promote the translocation of transcription factor EB (TFEB) into the nucleus [46,47]. In contrast, the glucosyltransferases in Legionella (Lgt1, Lgt2, and Lgt3) activate the mTORC1 pathway by inhibiting translation by targeting host elongation factor 1A [46]. In addition, a recent report illustrated an epigenetic mechanism that suppresses the host autophagic response mediated by the effector Lpg2936, which reduces the expression of autophagy-related genes ATG7 and LC3 during L. pneumophila infection [48]. Interestingly, L. pneumophila Dot/Icm substrate LegA9 appears to promote recognition of LCV for autophagy uptake and elimination via the induction of ubiquitin accumulation and association of the adaptor protein p62 on the vacuoles [49]. These previous observations revealed the extensive effector cohort and the sophisticated mechanisms that L. pneumophila has developed to manipulate host autophagy.
In this study, we demonstrated that, similar to most intracellular pathogens, such as S. Typhimurium and L. pneumophila, L. longbeachae also actively suppresses host autophagy during infection in a Dot/Icm-dependent manner. RavZ LLO in L. longbeachae is responsible for autophagy inhibitory activity and may act in the same manner as RavZ LP . Indeed, RavZ LLO and RavZ LP are functionally redundant, as the complement of L. pneumophila ΔravZ with RavZ LLO efficiently restores its capability to inhibit autophagy. This finding is consistent with an earlier observation that the deficiency in ER recruitment displayed by L. pneumophila ΔsidC/sdcA can be fully complemented by SidC LLO , albeit they share only 40% sequence identity [26]. The deletion of ravZ LLO did not affect the intracellular proliferation of L. longbeachae, suggesting the presence of other effector proteins that promote bacterial evasion of the host autophagy system. Comparative analysis of the effector repertoires revealed that orthologous proteins were found for RavZ, SidEs, Lpg2936, and Lpg1137 but not for Lgts, LpSpl, LegA9, and SetA in L. longbeachae (S5 Table), which is indicative of both potential similarities and distinctions between L. longbeachae and L. pneumophila in the modulation of autophagy.

PLOS ONE
Previous structural analyses of RavZ uncovered the similarity of its N-terminal domain with that in the Ulp family DUB-like enzymes [40]. Furthermore, coinfection of L. pneumophila and S. Typhimurium demonstrated the roles of RavZ in preventing ubiquitin recruitment to SCVs [42]. Paradoxically, RavZ cannot reduce the levels of ubiquitin on the LCVs in L. pneumophila-infected cells, possibly due to the presence of an antagonistic strategy used in LCVs to protect against RavZ [42]. These observations raise the possibility that, in addition to targeting LC3-PE, RavZ may possess DUB activity to deconjugate certain ubiquitinated substrates on SCVs. However, the DUB activity of RavZ was not established, as ectopic expression of RavZ did not affect cellular polyubiquitin levels, indicating an indirect role of RavZ in ubiquitin removal from SCVs [42]. Intriguingly, although it is not conclusive that RavZ LLO is a canonical DUB, ectopic expression of RavZ LLO in mammalian cells could decrease the levels of polyubiquitinated proteins through a mechanism that is independent of the catalytic cysteine essential for uncoupling LC3-PE. Consistently, RavZ LLO has been shown to regulate the association of polyubiquitinated species with LCVs during L. longbeachae and L. pneumophila infection. The ubiquitin feature of LCVs creates a signaling platform that promotes the recruitment of autophagy adaptors, including p62/SQSTM1, NDP52, NBR1, and optineurin [50]. Notably, changes in the level or nature of the polyubiquitin on the LCV reasonably affects adaptor recruitment. SidE family effector proteins can exclude adaptor proteins associated with the LCV, possibly by the generation of noncanonical ubiquitin linkages [51]. In contrast, LegA9 targets LCVs for autophagy uptake through increased ubiquitin labeling and p62/SQSTM1 recruitment of the LCV [49]. Hence, in addition to promoting the deconjugation of LC3-PE, RavZ LLO may facilitate autophagy evasion by reducing the levels of ubiquitinated proteins on the bacterial phagosome.